1. Field of the Invention
This invention relates to immunochemical assaying. Immunochemicalassays are proving of immense value in medicine and biology for the assaying of liquid samples, especially for example, body fluid samples such as blood or urine because of the sensitivity and specificity of such assays. The present invention is concerned with assaying for 3-ketosteroid compounds, notably .DELTA.4,3-ketosteroids and dihydrotestosterone. These compounds are mostly hormones, many of which are highly important to the proper functioning of the human body and include the sex hormones progesterone and testosterone as well as the well-known corticosteroid hormones aldosterone and cortisone. Accurate assay of these substances is of the utmost value in the diagnosis and treatment of many organic disorders.
In immunoassaying procedures, for a given target compound, a synthetic antigen is generally first prepared. Heretofore, this has usually been accomplished by coupling the target compound, through a coupling group to a carrier which confers antigenicity to the entire compound. The compound coupled to the carrier is usually known as a hapten and, when coupled, it functions as an antigenic determinant so that the antibodies produced will bind with the hapten. Thus, the antibodies produced should have a distinct and unique character, such that they will bind with only a specific compound or class of compounds. The objective in devising the synthetic hapten-carrier conjugate is to provide a compound which will generate antibodies that are specific to the target compound.
Antibodies are prepared by injecting the synthetic hapten-carrier conjugate into immunologically competent vertebrate animals, for example mammals, and recovering blood serum from them after they have had time to generate antibodies. Typical mammals are rabbits and goats.
The principal problem is usually that of producing sufficiently specific antibodies. Biological fluids such as blood and urine frequently contain very closely related compounds and it is common for antibodies to be unable to distinguish the target compound from close relatives, or sometimes even from distant ones. The antibody is then considered to be a poor one and is said to have low specificity and high cross-reactivity.
The assay itself is commonly a competitive binding assay. In a useful embodiment of such an assay, the target compound, which is not necessarily extracted, is allowed to compete with known quantities of a labeled standard to bind with a known quantity of specific antibody. From measurement of the proportion of the labeling in the standard-antibody complex that results, the amount of target compound present can be calculated. Radioactive isotope labeling is particularly convenient. Fluorescence perturbation and electron spin resonance have been used in the art. Normally it is necessary to remove any unreacted labeled standard, before making the determination on the antibody complex, although thereoretically, the determination could be made on the removed unreacted portion of the standard.
2. The Prior Art
Some thirty years ago, synthetic antigens were prepared by chemically coupling haptens to protein carriers. The antigens were administered to animals with a view to producing antibodies that would bind with the haptens. The objective was mostly to show binding. More recently the objective has been specificity of the antibody which is the quality of binding selectively with the desired compound and not with others.
Over the years there has been much research into different coupling methods and many synthetic antigens of this type have been proposed, described and prepared.
Coupling can proceed via the intermediary of a linking compound or compounds. At least two reactions are usually involved in one of which the hapten and linking compound are chemically coupled together and in the other of which the carrier and linking compound are coupled. There may be additional steps involving, for example, the coupling together of two linking compounds. It is usually desirable to couple a substantial plurality of haptens to a single carrier molecule. There are, however, known reaction sequences in which the hapten is coupled directly to the carrier in a single step, for example, by carbodiimide condensation of an amino group on one with a carboxyl group on the other to form a peptide or amide bond.